The nuclear factor-?B (NF-?B) family of dimeric transcription factors controls key biological events such as innate and immune responses, and cell proliferation and programmed cell death, by regulating the expression of a wide range of genes that are directly involved in those biological processes. The NF-?B family is comprised of five subunits, p50 (NF-kappaB1), p52 (NF-kappaB2), p65 (RelA), c-Rel and RelB, which form functional NF-kappaB dimers in a combinatorial manner. The p52/RelB heterodimer is one of the combinatorial dimers which is a key player transcription factor in lymphoid cells. p52 is derived from the precursor protein p100 through processing by the proteasome in response to specific stimuli. The mechanism of this unusual, signal-dependent partial degradation of a molecule by the proteasome is unknown. The focus of this application is to study how the signal-dependent processing of p 100 to p52 occurs. We propose a model to explain the mechanism that underlies this processing event. It is known that RelB does not associate with itself or with p65 and c-Rel. However, it preferentially associates with p100. These observations lead us to hypothesize that hetero-dimerization with RelB is a pre-requisite event for p100 to be processed into p52 and the formation of the p52/RelB heterodimer. This proposal will test whether or not association with RelB is an obligatory step for p100 processing, and whether the p100/RelB dimer is a structurally unique dimer compared to other NF-kappaB dimers. Our preliminary experiments have shown that in RelB deficient cells, p100 processing to p52 is blocked. We have further revealed that the dimerization domain of RelB is relatively unstable and this lack of stability might be critical for the formation of the inactive p100/RelB heterodimer. This proposal will integrate in vitro biochemical and X-ray crystallographic studies with in vivo experiments using various NF-kappaB subunit knock out cells to understand the mechanism of activation of an important NF-kappaB heterodimer.